The present invention relates to a method for simultaneous analysis of a saccharide mixture or, more particularly, to a method for the simultaneous analysis of a saccharide mixture including monosaccharides and oligosaccharides to be separated into individual constituents by means of the high-performance liquid chromatography as well as to an improved apparatus system used in the method.
As is known, conventional methods for the analysis of saccharides such as monosaccharides and oligosaccharides include the gel filtration method and normal-phase chromatographic method. It is a usual way in these methods that a differential refractometer is used for the detection of the component saccharides which do not emit fluorescence or do not exhibit absorption in the ultraviolet and visible ranges of light. Detection of monosaccharides and oligosaccharides is conducted also by using a differential refractometer.
The aforementioned analytical methods each have their respective disadvantages. For example, the gel filtration method is applicable to separation of monosaccharides from oligosaccharides but is not applicable to the separation between monosaccharides or between oligosaccharides having the same molecular weight without difficulties. In the normal-phase chromatographic method, the mobile phase is limited to have a single uniform composition, so that the method cannot be undertaken without sacrificing either completeness of elution of the oligosaccharides or separation among monosaccharides.
A further method is known for the simultaneous analysis of saccharides in which the sample mixture is subjected to a pre-treatment for conversion of the individual saccharides into their respective derivatives having characteristics for fluorescence emission or ultraviolet absorption followed by separation into the individual species by the high-performance liquid chromatographic method for detection by means of a fluorescence detector or ultraviolet absorption detector. This method is versatile in respect of the mobile phase which is not limited to a single composition but a plurality of mobile phases of different formulations can be employed for elution enabling separation among monosaccharides or among oligosaccharides to be advantageous as compared with the use of a single uniform mobile phase. This method, however, has a problem in the accuracy of the analytical results when the sample saccharide mixture contains a substance which influences on the yield of the reaction for conversion of a reducing saccharide into a derivative thereof. Accordingly, this method is not always satisfactory in respect of the quantitative accuracy when the method is applied to the high-sensitivity simultaneous analysis of reducing saccharides in a multicomponent sample such as foods.
Besides, a method is proposed in Japanese Patent Kokai 5-113439 and The Journal of Food Hygienic Society of Japan, volume 39, No. 5, pages 333-340 (1998), in which saccharides are separated by using two kinds or more of different mobile phases into component saccharides which are converted into their respective derivatives to be detected in a detector.
In these prior art methods, however, separation of the saccharides in an analytical column is followed by on-line admixing of the individual saccharides with a reagent for conversion of the same into their respective derivatives to be detected in a detector. It is accordingly unavoidable that the sample cells of the detector become stained during the analytical procedure by the reaction mixtures or the reagent solutions flowing through the cells resulting in an undesirable decrease in the detecting sensitivity, i.e. intensity of detectable fluorescence, as the number of the analytical runs increases.
It may be a natural countermeasure to solve the aforementioned problem of cell contamination to clean the detector cells after each of the analytical runs. This way of frequent cleaning of the detector cells, however, is not practical. Namely, cleaning of the detector cells must be undertaken in one of the following methods: (1) the analytical column is dismounted from the apparatus system and the pump is directly connected to the detector to introduce a cleaning solution to the cells, (2) the pump for mobile phase feeding is operated, without dismounting the analytical column, to feed the cleaning solvent; or (3) the cleaning solvent is fed by means of another pump connected to the analytical column at the outlet port of the eluate or downstream. These methods, however, have their respective disadvantages. For example, it is necessary in the first method (1) that the mobile-phase feed pump is turned off at least for a while for dismounting of the analytical column adversely affecting the accuracy for feed rate control and hence the accuracy of the analytical results. The second method (2) has a disadvantage that the cleaning solvent cannot be selected freely as limited by the material of the stationary phase filling the analytical column. The third method (3) has a problem that the cleaning solvent and the mobile phase are unavoidably intermixed more or less unless the mobile-phase feed pump is turned off during the cleaning treatment.
In addition, it is known that troubles are caused by a gas such as air originating in the solution or solvent used which stays in the pump to cause a decrease in the accuracy of flow rate control and hence a decrease in the reproducibility of the analytical results. It is sometimes the case that the air bubble enters the detector cell to disturb the performance of the detector. This problem is more serious when analysis is conducted continuously by using a mobile phase and a cleaning solution containing an organic solvent.